An IGFBP2-derived peptide promotes neuroplasticity and rescues deficits in a mouse model of Phelan-McDermid syndrome.

We developed an IGFBP2-mimetic peptide fragment, JB2, and showed that it promotes basal synaptic structural and functional plasticity in cultured neurons and mice. We demonstrate that JB2 directly binds to dendrites and synapses, and its biological activity involves NMDA receptor activation, gene transcription and translation, and IGF2 receptors. It is not IGF1 receptor-dependent. In neurons, JB2 induced extensive remodeling of the membrane phosphoproteome. Synapse and cytoskeletal regulation, autism spectrum disorder (ASD) risk factors, and a Shank3-associated protein network were significantly enriched among phosphorylated and dephosphorylated proteins. Haploinsufficiency of the SHANK3 gene on chromosome 22q13.3 often causes Phelan-McDermid Syndrome (PMS), a genetically defined form of autism with profound deficits in motor behavior, sensory processing, language, and cognitive function. We identified multiple disease-relevant phenotypes in a Shank3 heterozygous mouse and showed that JB2 rescued deficits in synaptic function and plasticity, learning and memory, ultrasonic vocalizations, and motor function; it also normalized neuronal excitability and seizure susceptibility. Notably, JB2 rescued deficits in the auditory evoked response latency, alpha peak frequency, and steady-state electroencephalography response, measures with direct translational value to human subjects. These data demonstrate that JB2 is a potent modulator of neuroplasticity with therapeutic potential for the treatment of PMS and ASD.

NYX-2925 Is a Novel N-Methyl-d-Aspartate Receptor Modulator that Induces Rapid and Long-Lasting Analgesia in Rat Models of Neuropathic Pain.

NYX-2925 [(2S,3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl)butanamide] is a novel N-methyl-d-aspartate (NMDA) receptor modulator that is currently being investigated in phase 2 clinical studies for the treatment of painful diabetic peripheral neuropathy and fibromyalgia. Previous studies demonstrated that NYX-2925 is a member of a novel class of NMDA receptor-specific modulators that affect synaptic plasticity processes associated with learning and memory. Studies here examined NYX-2925 administration in rat peripheral chronic constriction nerve injury (CCI) and streptozotocin-induced diabetic mechanical hypersensitivity. Additionally, NYX-2925 was examined in formalin-induced persistent pain model and the tail flick test of acute nociception. Oral administration of NYX-2925 resulted in rapid and long-lasting analgesia in both of the neuropathic pain models and formalin-induced persistent pain, but was ineffective in the tail flick model. The analgesic effects of NYX-2925 were blocked by the systemic administration of NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid. Microinjection of NYX-2925 into the medial prefrontal cortex of CCI rats resulted in analgesic effects similar to those observed following systemic administration, whereas intrathecal administration of NYX-2925 was ineffective. In CCI animals, NYX-2925 administration reversed deficits seen in a rat model of rough-and-tumble play. Thus, it appears that NYX-2925 may have therapeutic potential for the treatment of neuropathic pain, and the data presented here support the idea that NYX-2925 may act centrally to ameliorate pain and modulate negative affective states associated with chronic neuropathic pain.

Induction of Apoptosis in Metastatic Breast Cancer Cells: XV. Downregulation of DNA Polymerase-α - Helicase Complex (Replisomes) and Glyco-Genes.

In normal and cancer cells, successful cell division requires accurate duplication of chromosomal DNA. All cells require a multiprotein DNA duplication system (replisomes) for their existence. However, death of normal cells in our body occurs through the apoptotic process. During apoptotic process several crucial genes are downregulated with the upregulation of caspase pathways, leading to ultimate degradation of genomic DNA. In metastatic cancer cells (SKBR-3, MCF -7, and MDA-462), this process is inhibited to achieve immortality as well as overexpression of the enzymes for the synthesis of marker molecules. It is believed that the GSL of the lacto family such as LeX, SA-LeX, LeY, Lea, and Leb are markers on the human colon and breast cancer cells. Recently, we have characterized that a few apoptotic chemicals (cis-platin, L-PPMP, D-PDMP, GD3 ganglioside, GD1b ganglioside, betulinic acid, tamoxifen, and melphalan) in low doses kill metastatic breast cancer cells. The apoptosis-inducing agent (e.g., cis-platin) showed inhibition of DNA polymerase/helicase (part of the replisomes) and also modulated (positively) a few glycolipid-glycosyltransferase (GSL-GLTs) transcriptions in the early stages (within 2 h after treatment) of apoptosis. These Lc-family GSLs are also present on the surfaces of human breast and colon carcinoma cells. It is advantageous to deliver these apoptotic chemicals through the metastatic cell surfaces containing high concentration of marker glycolipids (Lc-GSLs). Targeted application of apoptotic chemicals (in micro scale) to kill the cancer cells would be an ideal way to inhibit the metastatic growth of both breast and colon cancer cells. It was observed in three different breast cancer lines (SKBR-3, MDA-468, and MCF-7) that in 2 h very little apoptotic process had started, but predominant biochemical changes (including inactivation of replisomes) started between 6 and 24 h of the drug treatments. The contents of replisomes (replisomal complexes) during induction of apoptosis are not known. It is known that DNA helicase activities (major proteins catalyze the melting of dsDNA strands) change during apoptotic induction process. Previously DNA Helicase-III was characterized as a component of the replication complexes isolated from carcinoma cells and normal rapid growing embryonic chicken brain cells. Helicase activities were assayed by a novel method (combined immunoprecipitation-ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of nicked "ACT-DNA," by determining values obtained from +/- aphidicolin added to the incubation mixtures. Very little is known about the stability of the "replication complexes" (or replisomes) during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during replication, repair, and recombination processes. In all three breast carcinoma cell lines (SKBR-3, MCF-7, and MDA-468), a common trend, decrease of activities of DNA polymerase-alpha and Helicase-III (estimated and detected with a polyclonal antibody), was observed, after cis-platin- and L-PPMP-induced apoptosis. Previously our laboratory has documented downregulation (within 24-48 h) of several GSL-GLTs with these apoptotic reagents in breast and colon cancer cells also. Perhaps induced apoptosis would improve the prognosis in metastatic breast and colon cancer patients.

The role of DNA methylation in ST6Gal1 expression in gliomas.

The mechanism of transcriptional silencing of ST6Gal1 in gliomas has not yet been elucidated. Multiple independent promoters govern the expression of the ST6Gal I gene. Here, we investigated whether epigenetic abnormalities involving DNA methylation affect ST6Gal1 expression. Transcript-specific qRT-PCR following exposure of glioma cell lines to 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, resulted in the re-expression of the normally quiescent ST6Gal1 mRNA driven exclusively by the P3 promoter sequence. The P3 promoter-specific transcription start site (TSS) was delineated by primer extension and core promoter sequences and associated functional transcription elements identified by deletion analysis utilizing chloramphenicol acetyltransferase reporter constructs. Minimal promoter activity was found to reside within the first 100 bp of the TSS and maximal activity was controlled by functional AP2 binding sites residing between 400 and 500 bp upstream of the initiation site. As altered AP2 binding was not directly associated with AP2 availability, these analyses demonstrate that ST6Gal1 transcription is regulated by DNA methylation within core promoter regions, ultimately by determining critical transcription factor accessibility within these regions. Transcriptional reactivation of ST6Gal1 expression by 5-aza-dC resulted in increased cell surface α2,6 sialoglycoconjugate expression, increased α2,6 sialylation of ß1 integrin, and decreased adhesion to fibronectin substrate: functional correlates of decreased invasivity. The effects of global hypomethylation are not glycome-wide. Focused glycotranscriptomic analyses of three invasive glioma cell lines following 5-aza-dC treatment demonstrated the modulation of select glycogene transcripts. Taken together, these results demonstrate that epigenetic modulation of ST6Gal1 expression plays a key role in the glioma phenotype in vitro and that that therapeutic approaches targeting elements of the epigenetic machinery for the treatment of human glioblastoma are warranted.

Integrated Transcriptomic and Glycomic Profiling of Glioma Stem Cell Xenografts.

Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) have the innate ability to migrate or home toward and engraft in tumors such as glioblastoma (GBM). Because of this unique property of BM-hMSCs, we have explored their use for cell-mediated therapeutic delivery for the advancement of GBM treatment. Extravasation, the process by which blood-borne cells­such as BM-hMSCs­enter the tissue, is a highly complex process but is heavily dependent upon glycosylation for glycan-glycan and glycan-protein adhesion between the cell and endothelium. However, in a translationally significant preclinical glioma stem cell xenograft (GSCX) model of GBM, BM-hMSCs demonstrate unequal tropism toward these tumors. We hypothesized that there may be differences in the glycan compositions between the GSCXs that elicit homing ("attractors") and those that do not ("non-attractors") that facilitate or impede the engraftment of BM-hMSCs in the tumor. In this study, glycotranscriptomic analysis revealed significant heterogeneity within the attractor phenotype and the enrichment of high mannose type N-glycan biosynthesis in the non-attractor phenotype. Orthogonal validation with topical PNGase F deglycosylation on the tumor regions of xenograft tissue, followed by nLC-ESI-MS, confirmed the presence of increased high mannose type N-glycans in the non-attractors. Additional evidence provided by our glycomic study revealed the prevalence of terminal sialic acid-containing N-glycans in non-attractors and terminal galactose and N-acetyl-glucosamine N-glycans in attractors. Our results provide the first evidence for differential glycomic profiles in attractor and non-attractor GSCXs and extend the scope of molecular determinates in BM-hMSC homing to glioma.

Use of ENCODE resources to characterize novel proteoforms and missing proteins in the human proteome.

We describe the utility of integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the "missing proteins", novel proteoforms, and PTMs. On one hand, databases in combination with bioinformatic tools are efficiently utilized to establish microarray-based transcript analysis and supply rapid protein identifications in clinical samples. On the other hand, sequence libraries are the foundation of targeted protein identification and quantification using mass spectrometric and immunoaffinity techniques. The results from combining proteoENCODEdb searches with experimental mass spectral data indicate that some alternative splicing forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research.

Integrative biological analysis for neuropsychopharmacology.

Although advances in psychotherapy have been made in recent years, drug discovery for brain diseases such as schizophrenia and mood disorders has stagnated. The need for new biomarkers and validated therapeutic targets in the field of neuropsychopharmacology is widely unmet. The brain is the most complex part of human anatomy from the standpoint of number and types of cells, their interconnections, and circuitry. To better meet patient needs, improved methods to approach brain studies by understanding functional networks that interact with the genome are being developed. The integrated biological approaches--proteomics, transcriptomics, metabolomics, and glycomics--have a strong record in several areas of biomedicine, including neurochemistry and neuro-oncology. Published applications of an integrated approach to projects of neurological, psychiatric, and pharmacological natures are still few but show promise to provide deep biological knowledge derived from cells, animal models, and clinical materials. Future studies that yield insights based on integrated analyses promise to deliver new therapeutic targets and biomarkers for personalized medicine.

Diabetes changes expression of genes related to glutamate neurotransmission and transport in the Long-Evans rat retina.

PURPOSE: This study investigated changes in the transcript levels of genes related to glutamate neurotransmission and transport as diabetes progresses in the Long-Evans rat retina. Transcript levels of vascular endothelial growth factor (VEGF), erythropoietin, and insulin-like growth factor binding protein 3 (IGFBP3) were also measured due to their protective effects on the retinal vasculature and neurons. METHODS: Diabetes was induced in Long-Evans rats with a single intraperitoneal (IP) injection of streptozotocin (STZ; 65 mg/kg) in sodium citrate buffer. Rats with blood glucose >300 mg/dl were deemed diabetic. Age-matched controls received a single IP injection of sodium citrate buffer only. The retinas were dissected at 4 and 12 weeks after induction of diabetes, and mRNA and protein were extracted from the left and right retinas of each rat, respectively. Gene expression was analyzed using quantitative real-time reverse-transcription PCR. Enzyme-linked immunosorbent assay was used to quantify the concentration of VEGF protein in each retina. Statistical significance was determined using 2×2 analysis of variance followed by post-hoc analysis using Fisher's protected least squares difference. RESULTS: Transcript levels of two ionotropic glutamate receptor subunits and one glutamate transporter increased after 4 weeks of diabetes. In contrast, 12 weeks of diabetes decreased the transcript levels of several genes, including two glutamate transporters, four out of five N-methyl-D-aspartate (NMDA) receptor subunits, and all five kainate receptor subunits. Diabetes had a greater effect on gene expression of NMDA and kainate receptor subunits than on the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits, for which only GRIA4 significantly decreased after 12 weeks. VEGF protein levels were significantly increased in 4-week diabetic rats compared to age-matched control rats whereas the increase was not significant after 12 weeks. Transcript levels of VEGF and VEGF receptors were unchanged with diabetes. Erythropoietin and IGFBP3 mRNA levels significantly increased at both time points, and IGFBP2 mRNA levels increased after 12 weeks. CONCLUSIONS: Diabetes caused significant changes in the transcriptional expression of genes related to ionotropic glutamate neurotransmission, especially after 12 weeks. Most genes with decreased transcript levels after 12 weeks were expressed by retinal ganglion cells, which include glutamate transporters and ionotropic glutamate receptors. Two genes expressed by retinal ganglion cells but unrelated to glutamate neurotransmission, γ-synuclein (SNCG) and adenosine A1 receptor (ADORA1), also had decreased mRNA expression after 12 weeks. These findings may indicate ganglion cells were lost as diabetes progressed in the retina. Decreased expression of the glutamate transporter SLC1A3 would lead to decreased removal of glutamate from the extracellular space, suggesting that diabetes impairs this function of Müller cells. These findings suggest that ganglion cells were lost due to glutamate excitotoxicity. The changes at 12 weeks occurred without significant changes in retinal VEGF protein or mRNA, although higher VEGF protein levels at 4 weeks may be an early protective response. Increased transcript levels of erythropoietin and IGFBP3 may also be a protective response.

Chromosome 19 annotations with disease speciation: a first report from the Global Research Consortium.

A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented ( http://www.c-hpp.org ). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC-MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks.

Ganglioside biosynthesis in developing brains and apoptotic cancer cells: X. regulation of glyco-genes involved in GD3 and Sialyl-Lex/a syntheses.

Gangliosides, the acidic glycosphingolipids (GSLs) containing N-acetylgalactosamine and sialic acid are ubiquitous in the central nervous system. At least six DSL-glycosyltransferase activities (GLTs Gangliosides, the acidic glycosphingolipids (GSLs) containing N-acetylgalactosamine and sialic acid (or NAc-Neuraminic acid) are ubiquitous in the central nervous system. At least six GSL-glycosyltransferase activities (GLTs) of Basu-Roseman pathway catalyzing the biosynthesis of these gangliosides have been characterized in developing chicken brains. Most of these glyco-genes are expressed in the early stages (7-17 days) of brain development and lowered in the adult stage, but the cause of reduction of enzymatic activities of these GLTs in the adult stages is not known. In order to study glyco-gene regulation we used four clonal metastatic cancer cells of colon and breast cancer tissue origin (Colo-205, SKBR-3, MDA-468, and MCF-3). The glyco-genes for synthesis of SA-LeX and SA-LeA (which contain N-acetylglucosamine, sialic acid and fucose) in these cells were modulated differently at different phases (between 2 and 48 h) of apoptotic inductions. L-PPMP, D-PDMP (inhibitor of glucosylceramide biosynthesis), Betulinic Acid (a triterpinoid isolated from bark of certain trees and used for cancer treatment in China), Tamoxifen a drug in use in the west for treatment of early stages of the disease in breast cancer patients), and cis-platin (an inhibitor of DNA biosynthesis used for testicular cancer patients) were used for induction of apoptosis in the above-mentioned cell lines. Within 2-6 h, transcriptional modulation of a number of glyco-genes was observed by DNA-micro-array (containing over 300 glyco genes attached to the glass cover slips) studies. Under long incubation time (24-48 h) almost all of the glyco-genes were downregulated. The cause of these glyco-gene regulations during apoptotic induction in metastatic carcinoma cells is unknown and needs future investigations for further explanations. These apoptotic agents could be employed as a new generation of anti-cancer drugs after properly delivered to the patients.

Overexpression of ST6GalNAcV, a ganglioside-specific alpha2,6-sialyltransferase, inhibits glioma growth in vivo.

Aberrant cell-surface glycosylation patterns are present on virtually all tumors and have been linked to tumor progression, metastasis, and invasivity. We have shown that expressing a normally quiescent, glycoprotein-specific alpha2,6-sialyltransferase (ST6Gal1) gene in gliomas inhibited invasivity in vitro and tumor formation in vivo. To identify other glycogene targets with therapeutic potential, we created a focused 45-mer oligonucleotide microarray platform representing all of the cloned human glycotranscriptome and examined the glycogene expression profiles of 10 normal human brain specimens, 10 malignant gliomas, and 7 human glioma cell lines. Among the many significant changes in glycogene expression observed, of particular interest was the observation that an additional alpha2,6-sialyltransferase, ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha2,6-sialyltransferase 5 (ST6GalNAcV), was expressed at very low levels in all glioma and glioma cell lines examined compared with normal brain. ST6GalNAcV catalyzes the formation of the terminal alpha2,6-sialic acid linkages on gangliosides. Stable transfection of ST6GalNAcV into U373MG glioma cells produced (i) no change in alpha2,6-linked sialic acid-containing glycoproteins, (ii) increased expression of GM2alpha and GM3 gangliosides and decreased expression of GM1b, Gb3, and Gb4, (iii) marked inhibition of in vitro invasivity, (iv) modified cellular adhesion to fibronectin and laminin, (v) increased adhesion-mediated protein tyrosine phosphorylation of HSPA8, and (vi) inhibition of tumor growth in vivo. These results strongly suggest that modulation of the synthesis of specific glioma cell-surface glycosphingolipids alters invasivity in a manner that may have significant therapeutic potential.

Glycomic and transcriptomic response of GSC11 glioblastoma stem cells to STAT3 phosphorylation inhibition and serum-induced differentiation.

A glioblastoma stem cell (GSC) line, GSC11, grows as neurospheres in serum-free media supplemented with EGF (epidermal growth factor) and bFGF (basic fibroblast growth factor), and, if implanted in nude mice brains, will recapitulate high-grade glial tumors. Treatment with a STAT3 (signal transducer and activator of transcription 3) phosphorylation inhibitor (WP1193) or 10% FBS (fetal bovine serum) both led to a decrease in expression of the stem cell marker CD133 in GSC11 cells, but differed in phenotype changes. Altered glycolipid profiles were associated with some differentially expressed glycogenes. In serum treated cells, an overall increase in glycosphingolipids may be due to increased expression of ST6GALNAC2, a sialyltransferase. Serum treated cells express more phosphatidylcholine (PC), short chain sphingomyelin (SM) and unsaturated long chain phosphatidylinositol (PI). Decrease of a few glycosphingolipids in the STAT3 phosphorylation inhibited cells may be linked to decreased transcripts of ST6GALNAC2 and UGCGL2, a glucosylceramide synthase. A rare 3-sulfoglucuronylparagloboside carrying HNK1 (human natural killer-1) epitope was found expressed in the GSC11 and the phenotypically differentiated cells. Its up-regulation correlates with increased transcripts of a HNK1 biosynthesis gene, B3GAT2 after serum treatment. Taken together with a quantitative phosphoproteomic study of the same GSC line (C. L. Nilsson, et al. J. Proteome Res. 2010, 9, 430-443), this report represents the most complete systems biology study of cancer stem cell (CSC) differentiation to date. The synergies derived by the combination of glycomic, transcriptomic and phosphoproteomic data may aid our understanding of intracellular and cell-surface events associated with CSC differentiation.

Polar lipid remodeling and increased sulfatide expression are associated with the glioma therapeutic candidates, wild type p53 elevation and the topoisomerase-1 inhibitor, irinotecan.

We report changes in gene and polar lipid expression induced by adenovirus-delivered wild-type (wt) p53 gene and chemotherapy of U87 MG glioblastoma cells, a treatment known to trigger apoptosis and cell cycle arrest. Sulfatides (sulfonated glycolipids) were most highly modulated by wild-type p53 treatment; however, no changes were observed in expression levels of mRNA for genes involved in sulfatide metabolism, indicating post-transcriptional control of sulfatide synthesis. Modulation of the aglycones of GD1 and GM1b was observed in wild-type p53-treated cells. The treatment also leads to an increase in phospholipids such as phosphatidyl inositols, phosphatidyl serines, phosphatidyl glycerols, and phosphatidyl ethanolamines, especially hydroxylated phospholipids. These dramatic changes in the composition of cellular glycolipids in response to p53 gene expression and cytotoxic chemotherapy treatment indicate the large role that they play in cell signaling. The use of the human glioma cell line U87 appears to be an excellent model system both in tissue culture and in intracranial murine xenograft models to further characterize the role of sulfatides in modulating glioma responsivity to therapeutic agents.

The glycobiology of brain tumors: disease relevance and therapeutic potential.

The oligosaccharides that decorate cell surface glycoconjugates play important roles in intercellular recognition and cell-extracellular matrix interactions, and thus the regulation of cellular migration, metastasis and invasivity. Virtually all tumor cells display aberrant cell-surface glycosylation patterns brought about by alterations in their biosynthetic machinery. This holds true for highly invasive, malignant brain tumors as well as tumor cells that metastasize to the brain. The field of glycobiology is well established with essentially all of the biochemical pathways for oligosaccharide metabolism characterized and all of the 'glycogenes' involved in these pathways cloned. Yet there has been a paucity of progress toward the development of therapeutics. However, recent studies aimed at controlled glycosylation of therapeutic antibodies and mucins with anticancer vaccine potential, the emergence of new and highly sensitive tools for the identification of tumor-associated biomarkers and the manipulation of the expression of glycogenes that inhibit brain tumor invasivity have emerged. The opportunity now exists to answer questions as to how glycogenes are regulated at the genomic and transcriptomic level and how altered glycogene expression patterns lead to altered cell surface glycoconjugates. These studies should lead to the development of ways to directly regulate tumor cell glycogene expression, which should have significant therapeutic potential.

Post-translational and transcriptional regulation of glycolipid glycosyltransferase genes in apoptotic breast carcinoma cells: VII. Studied by DNA-microarray after treatment with L-PPMP.

Functions of glycosphingolipids on the eukaryotic cell membranes during the onset of oncogenic processes and cell death are not well understood. Inhibitors of glycosphingolipid biosynthesis were recently found to trigger apoptosis in many carcinoma including breast cancer SKBR-3, MCF-7, and MDA-468 cells through either intrinsic or extrinsic apoptotic pathways as we previously reported. These anti-cancer inhibitors could increase ceramide concentration by blocking functions of glycolipid glycosyltransferases (GLTs). In this study, using a novel fluorescent dye (ASK-0) revealed the damage of cell organelle membranes by an inhibitor of glucosylceramide biosynthesis (L-PPMP). A highly drug- and cell-dependent regulation of MAPKs was also found by cis-platin and L-PPMP when inducing apoptosis in SKBR-3, MCF-7, and MDA-468 cells. A dose and time-dependent regulation of GLTs were investigated by enzymatic assay and DNA microarray analyses. These GLTs are involved in biosynthesis of Le(X) and sialosyl-Le(X) (neolactosyl-ceramide series) such as GalT-4 (UDP-Gal: LcOse3cer beta-galactosyltransferase, GalT-5 (UDP-Gal: nLcOse4Cer alpha1, 3galactosyltransferase, FucT-3 (GDP-Fucose: LM1 alpha1, 4fucosyltransferase). A similar effect was observed with the GLTs involved in the biosyntheses of Gg-series gangliosides, such as SAT-4 (CMP-NeuAc: GgOse4Cer alpha2, 3sialyltransferase, and SAT-2 (CMP-NeuAc: GM3 alpha2, 8sialyltransferase). The glycol-related gene DNA-microarrays also suggested the transcriptional regulation of several GLTs involved in the biosynthesis of neolactosylceramide containing cell-surface antigens in these apoptotic breast carcinoma cells. In the early apoptotic stages (2 to 6 h after L-PPMP treatment) in addition to GlcT-1 gene, several genes (betaGalTs and betaGlcNAcTs) in the SA-Le(a) pathway were stimulated.

Focused microarray analysis of glyco-gene expression in human glioblastomas.

Altered glycosylation has been linked to cancer cell metastasis and invasivity. We have previously shown that expressing a specific sialyltransferase gene in gliomas inhibited tumor formation, in vivo. In order to identify other "glyco-gene" targets with therapeutic potential, focused 45-mer oligonucleotide microarrays were constructed containing all of the cloned human glyco-genes. Gene expression profiles of normal human brain and malignant gliomas were compared and microarray datasets analyzed using significance analysis of microarrays algorithms. There were 11 genes more highly expressed in gliomas compared to normal brain and 25 genes more highly expressed in normal brain compared to gliomas. Among the most noteworthy were high levels of MAN2A2 and ST6GalNAcV in normal brain tissue and high levels of POFUT1 and CHI3L1 in the gliomas, all changes corroborated by qRT-PCR. Historically, identification of changes in tumor-associated glycoconjugate expression was obtained by measuring individual enzyme activities or structural changes of specific molecules. With microarray technology, it is possible to measure all of genes associated with glycoconjugate biosynthesis and degradation simultaneously. Our data demonstrate that there are many significant and novel differences in glyco-gene expression that represent potential targets for the development of therapeutics for the treatment of brain tumors.

Cellular thiol pools are responsible for sequestration of cytotoxic reactive aldehydes: central role of free cysteine and cysteamine.

Cellular thiol pools have been shown to be important in the regulation of the redox status of cells, providing a large antioxidant pool consisting of free thiols, thiols bound in the disulfide form and thiols bound to proteins. However, experimental studies with the thiol cysteamine and its disulfide cystamine have demonstrated dramatic cytoprotection in experimental models where antioxidants provide only minor protection. These data suggest that an alternate action of thiols is important in their cytoprotective actions. A common feature of the in vitro and in vivo models, where these thiol agents demonstrate cytoprotection, is the generation of cytotoxic aldehydes. We therefore studied the actions of cystamine, cysteamine and several reference thiol agents as cytoprotectants against cell death induced by increased "aldehyde load". We found that all the thiol agents examined provided dramatic protection against aldehyde-induced cell death in SN56 cholinergic neurons, under conditions in which acrolein induced 100% cell death. With regard to mechanism of action, the reference thiols cysteine, N-acetylcysteine, 2-mercaptoethanesulfonic acid, mercapto-propionyglycine, and cysteamine can directly sequester aldehydes. In addition, these thiols were all found to augment intracellular cysteine levels via disulfide interchange reactions. Cysteamine and cystamine also augmented basal intracellular cysteamine levels. Our data, for the first time, demonstrate the importance of intracellular thiols in sequestering toxic reactive aldehyde products of lipid peroxidation and polyamine metabolism. In addition it appears that pharmacological manipulation of intracellular thiol pools might offer a new approach in the design of neuroprotective drug candidates.

Neurochemical analysis of amino acids, polyamines and carboxylic acids: GC-MS quantitation of tBDMS derivatives using ammonia positive chemical ionization.

The GC-MS quantitation of a large number of neurochemicals utilizing a single derivatization step is not common but is provided by the reagent N-(tert-butyldimethylsilyl)-N-methyltrifluro-acetamide (MTBSTFA). Previous workers have utilized this derivative for GC-MS analyses of amino acids, carboxylic acids and urea with electron impact (EI) and with positive chemical ionization (PCI; methane as reagent gas). However, these conditions yield significant fragmentation, decreasing sensitivity and in some cases reducing specificity for quantitation with selected ion monitoring (SIM). Additionally, the majority of studies have used a single internal standard to quantitate many compounds. In this study we demonstrate that using isotopic dilution combined with ammonia as the reagent gas for PCI analyses, results in high precision and sensitivity in analyzing complex neurochemical mixes. We also demonstrate for the first time the utility of this derivative for the analysis of brain polyamines and the dipeptide cysteinyl glycine. In the case of ammonia as the reagent gas, all amino acids, polyamines and urea yielded strong [MH](+) ions with little or no fragmentation. In the case of carboxylic acids, [M+18](+) ions predominated but [MH](+) ions were also noted. This approach was used to analyze superfusates from hippocampal brain slices and brain tissue extracts from brain lesion studies. The advantages of this methodology include: (i) simple sample preparation; (ii) a single derivatization step; (iii) direct GC-MS analysis of the reaction mix; (iv) high precision as a result of isotopic dilution analyses; (v) high sensitivity and specificity as a result of strong [MH](+) ions with ammonia reagent gas; (vi) no hydrolysis of glutamine to glutamate or asparagine to aspartate; and (vii) applicability to a wide range of neurochemicals.